我们承认奥斯汀Cyphersmith和Mayandi Sivaguru（卡尔R. Woese研究所基因组生物学，伊利诺伊大学厄巴纳 - 香槟分校），与显微镜援助和在显微镜核心慷慨地容纳屏和视频捕捉。

作者宣称，他们没有竞争的经济利益。

在我们的实验中，我们已经发现，最常见的故障都涉及到制造的阵列的质量和差的特征在于在感兴趣的生物系统响应。我们建议读者参考表1在细胞微阵列实验常见失效模式和相关的故障排除步骤。特别是关于阵列的质量，我们推荐以下。确认排列程序，参数，以及使用荧光标记的分子，例如若丹明共轭葡聚糖缓冲器的技术质量和鲁棒性。彻底清洁针之前或按照制造商的说明书排列，并进一步在视觉上后检查销渠道使用光学显微镜是明确的碎片。使用通用的蛋白质染色或免疫标记确认阵列生物分子的保留。需要注意的是低于70 kDa的分子量的生物分子经常不保留在水凝胶23， </<html> SUP&gt; 31。使用多种细胞类型验证阵列的生物分子的细胞的功能。请注意，只有贴壁细胞与阵列兼容;另外，粘附到阵列是依赖于两个小区特定属性（ 例如 ，整联表达谱），并将选定ECM蛋白。 由于篇幅有限，我们这里不提供阵列设计，布局和制造广泛的治疗和读者参考以前的作品23，25。我们一般使用10-20独特的生物分子的条件（ 即 ，5-10个/条件）组成的100点的子阵列（150微米的光斑直径，450微米的中心-至-中心距离）。子阵列的一个阵列的数量取决于所感兴趣的生物分子的条件的数量，这可以舒适地按比例放大到1,280上的一个25×75毫米的显微镜载玻片（〜6400点的64子阵列）外部参照"&gt; 25,31以上参数将取决于所感兴趣的图案尺寸进一步变化;能够从75产生图案的销- 450微米都是现成的。 阵列实验最好使用其他文化形式的利息高得分排列的情况下，法显装置和生物模型系统验证补充。具体而言，我们建议使用批量培养符合标准的分子生物学技术（ 例如 ，定量RT-PCR，免疫印迹）或标准TFM一起进一步验证的选择摆着条件的影响。遗传操作在一个适当的生物模型系统还可以用来确认在阵列观察效果感兴趣的因子（ 例如 ，敲除或过表达）。 在体内动物模型表示验证的另一种手段和最近使用的，例如，以确认在半乳凝素-3和半乳凝素8的中心作用肺癌转移的利基，如通过细胞芯片31，49初步确定。 其他一些方法已被用来探测细胞功能的微环境调控，包括各种二维微加工系统18，50，51，52，53，54，55和三维设计的生物材料系统56，57，58 ，59，60，61。与其他方法相比，这里描述的细胞微阵列平台的特别的优点包括:（1）的吞吐量达到数百或数千的因素的不同组合，可实现的相互作用效应的分析; （2）访问，自动成像和分析; （3）与排列因素控制介绍生物化学和生物物理读数的整合; （4），以改变基底材料特性的能力;和（5）含量高的细胞命运和功能的单细胞分析。 综上所述，随着TFM细胞芯片的可调谐基板刚性基材的结合使双方生物化学和生物物理线索的深入刻画。如这里介绍的，该平台是一般化并可以容易地应用于各种贴壁细胞类型和组织的上下文向细胞分化和机械传导的组合微环境调节的改进的理解。

细胞和周围微环境因素之间的相互作用介导了大量的各种整个发育，稳态和疾病的发病机制1，2，3，4的生物过程。这些微环境相互作用包括通过配体 - 受体结合递送的可溶性因子的细胞，细胞 - 基质结合，和细胞 - 细胞相互作用。除了上述的生化考虑，生物物理参数，如底物的机械性能（ 例如 ，杨氏模量，孔隙率）和电池的形状，和相关的下游机械传导日益得到认可作为细胞分化5，6，7，8的关键介质， 9 <sup&gt; 10。从这些微环境相互作用产生的信号作为输入，基因网络和信号通路。此外，这些细胞的固有组分也通过分泌的因子和基质降解酶，完成细胞内在遗传计划和细胞外微环境因素5，11，12之间的复合物共同监管环路反馈提供给微环境。 使用用于微环境因素的控制介绍工程系统的范围内不同的上下文13，14，15的已被证明是有用的。特别是微制造的系统已通过小型化13促进蛋白质和细胞以及高度并行分析的精确的空间图案化<sup类="外部参照"&gt; 16，17，18，19，20，21，22。细胞微阵列表示一个这样的微加工系统，其中的生物分子的组合是接触印刷到弹性聚丙烯酰胺水凝胶基底23，24，25。细胞粘接性成分（即基质蛋白）的加入使芯片上的持续细胞粘附和文化，这是经常其次是通过免疫组化和荧光记者下游分析。细胞芯片已经富有成效针对实现肝细胞表型的23，26，神经前体分化27 mammar的改进认识ý祖命运决定28，胚胎干细胞的维持/分化23，29，30，肺癌转移31，以及在黑色素瘤32的治疗反应。我们最近证明了使用细胞微阵列的用于限定在定形内胚层规范33，肝脏祖细胞分化34，35，和肺肿瘤细胞对药物的反应36外基质（ECM）蛋白的组合物的作用。在这些作品中，我们把重点放在扩大阵列平台的组合能力和探索细胞内在信号与细胞外基质成分及生物力学的交叉点。此外，我们在这个阵列平台上实现生物物理读数能力提供定量CHARAC细胞收缩的分化terize作用处理35。要做到这一点，我们综合牵引力显微镜（TFM）与细胞芯片，使细胞产生牵引的高通量评估。 TFM是测量细胞产生的牵引力一种广泛使用的方法，并提供了有关单细胞和组织水平的功能与组合物和局部微环境37，38，39，40的生物力学的协调显著见解。因此，TFM与细胞微阵列结合提供了一个高通量系统，用于测量键，生理学相关的生物物理参数。 这里描述的细胞微阵列平台由四部分组成:聚丙烯酰胺基板，阵列，细胞培养和测定的读出的制造，以及数据分析的制造。看到图1为前三个实验部分的示意性概要;参见图2的最后一节的重点放在免疫荧光分析数据的概要总结。为了适应在细胞微阵列平台来生物力学细胞-基底相互作用的研究中，我们使用可调谐的杨氏模量，但相似的孔隙率的聚丙烯酰胺底物，每Wen 等。 41。以使由它们的底物对细胞施加的力的TFM测量，我们实现除了厚的玻璃显微镜载片经常被其它基团使用的玻璃底培养皿的格式。因此，这种细胞芯片平台能够细胞分化通过对显微镜载玻片和细胞产生的力通过免疫荧光TFM单独的玻璃底菜并行测量。我们也应用了一些改进通常与电池芯片使用的分析方法。 SpecificalLY，代替整体岛强度的参数的Z得分，我们测量的单细胞的强度和以占非正常分布和更准确地描述细胞行为申请位数正常化。我们相信，这些改进提供了走向其生物化学和生物物理线索发挥显著，交叉作用的生物学过程的调查特别有用。此外，我们的分析改进使细胞芯片的一系列对这些单细胞和群体水平的行为发散细胞功能研究中的应用。

<table><tbody><tr><td>0.2 &amp;micro;m syringe filter</td><td>Pall Corporation</td><td>4433</td><td>Match with appropriately-sized Luer lock plastic syringes.</td></tr><tr><td>100 &amp;times; penicillin&amp;ndash;streptomycin solution</td><td>Fisher Scientific</td><td>SV30010</td><td></td></tr><tr><td>22 &amp;times; 60 mm coverglasses</td><td>Electron Microscopy Sciences</td><td>63765</td><td></td></tr><tr><td>3-(trimethoxysilyl)propyl methacrylate (3-TPM)</td><td>Sigma-Aldrich</td><td>440159</td><td>Store under inert gas per manufacturer&#039;s instructions. Exposure of 3-TPM to air could compromise silanization of glass substrates. CAUTION: 3-TPM is a combustible liquid. Keep away from heat, sparks, open flames, and hot surfaces and use only in a chemical fume hood.</td></tr><tr><td>3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS)</td><td>Sigma-Aldrich</td><td>C3023</td><td></td></tr><tr><td>35 mm glass-bottom Petri dishes</td><td>Cell E&amp;amp;G</td><td>GBD00002-200</td><td>13 mm well consisting of #1.5 coverglass. Enables TFM and live-cell imaging.</td></tr><tr><td>384-well polypropylene V-bottom microplate, non-sterile</td><td>USA Scientific</td><td>1823-8400</td><td></td></tr><tr><td>6-well polystyrene microplates</td><td>Fisher Scientific</td><td>08-772-1B</td><td>35 mm glass-bottom Petri dishes fit into wells of microplate, easing array fabrication.</td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>179973</td><td></td></tr><tr><td>Acrylamide</td><td>Sigma-Aldrich</td><td>A3553</td><td>CAUTION: Exposure to acrylamide can result in acute toxicity and irritation. Wear protective gloves, clothing, and eye protection.</td></tr><tr><td>Collagen I, rat tail</td><td>EMD Millipore</td><td>08-115MI</td><td></td></tr><tr><td>Collagen III, human</td><td>EMD Millipore</td><td>CC054</td><td></td></tr><tr><td>Collagen IV, human</td><td>EMD Millipore</td><td>CC076</td><td></td></tr><tr><td>Crosslinker, 365 nm</td><td>UVP</td><td>CL-1000</td><td></td></tr><tr><td>Dextran, rhodamine B-conjugated, 70 kDa</td><td>ThermoFisher Scientific</td><td>D1841</td><td>Used as a marker for array location.</td></tr><tr><td>Dimethyl sulfoxide</td><td>Fisher Scientific</td><td>BP231</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (PBS)</td><td>Fisher Scientific (HyClone)</td><td>SH3001302</td><td></td></tr><tr><td>Ethyl alcohol</td><td>Decon Labs</td><td>2701</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>ED</td><td></td></tr><tr><td>Fc-recombinant DLL1, mouse</td><td>R&amp;amp;D Systems</td><td>5026-DL-050</td><td></td></tr><tr><td>Fc-recombinant DLL4, mouse</td><td>AdipoGen</td><td>AG-40A-0145-C050</td><td></td></tr><tr><td>Fc-recombinant JAG1, rat</td><td>R&amp;amp;D Systems</td><td>599-JG-100</td><td></td></tr><tr><td>Fibronectin, human</td><td>Sigma-Aldrich</td><td>F2006</td><td></td></tr><tr><td>Fluorescent microscope, inverted</td><td>Zeiss</td><td>Axiovert 200M</td><td>Ensure microscope is equipped with a robotic stage for both automated fluorescent imaging and TFM. Environmental control (&lt;em&gt;i.e&lt;/em&gt;., 37 &amp;deg;C and 5% CO&lt;sub&gt;2&lt;/sub&gt;) is highly advisable for TFM.</td></tr><tr><td>Fluoromount G with DAPI</td><td>SouthernBiotech</td><td>0100-20</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Sigma-Aldrich</td><td>695092</td><td>CAUTION: Acetic acid is flammable and corrosive. Wear protective gloves, clothing, and eye protection.</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>M6145</td><td></td></tr><tr><td>Irgacure 2959</td><td>BASF Corporation</td><td>55047962</td><td></td></tr><tr><td>Laminin, mouse</td><td>EMD Millipore</td><td>CC095</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>179957</td><td></td></tr><tr><td>Microarray scanner</td><td>GenePix</td><td>4000B</td><td>Fluorophores must be Cy3- or Cy5-compatible.</td></tr><tr><td>Microarrayer</td><td>Digilab</td><td>OmniGrid Micro</td><td>Other microarrayers of similar or greater capability can readily be substituted.</td></tr><tr><td>Microscope slides, 25 &amp;times; 75 mm</td><td>Sigma-Aldrich</td><td>CLS294775X25</td><td>~0.9 &amp;ndash; 1.1 mm thickness.</td></tr><tr><td>&lt;em&gt;N,N&amp;prime;&lt;/em&gt;-Methylenebisacrylamide (bisacrylamide)</td><td>Sigma-Aldrich</td><td>M7279</td><td>CAUTION: Exposure to acrylamide can result in acute toxicity and irritation. Wear protective gloves, clothing, and eye protection.</td></tr><tr><td>Paraformaldehyde (PFA), 16% v/v</td><td>Electron Microscopy Sciences</td><td>RT15710</td><td>Prepare PFA fresh (do not store) for optimal fixation. CAUTION: Exposure to PFA can result in acute toxicity and can also irritate or corrode skin on contact. Wear protective gloves, clothing, and eye protection and use only in a chemical fume hood.</td></tr><tr><td>Protein A/G, recombinant</td><td>ThermoFisher Scientific</td><td>21186</td><td></td></tr><tr><td>Pyrex drying tray, 2,000&amp;nbsp;mL</td><td>Fisher Scientific</td><td>15-242B</td><td></td></tr><tr><td>Rectangular 4-chambered culture dish</td><td>Fisher Scientific (Nunc)</td><td>12-565-495</td><td>For cell culture on arrayed microscope slides.</td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>S2889</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>415413</td><td>CAUTION: NaOH is highly caustic and can cause severe skin burns and eye damage. Wear protective gloves, clothing, and eye protection.</td></tr><tr><td>Stealth pin for arraying</td><td>ArrayIt</td><td>SMP3</td><td>Clean pins after each array run using the instructions of the manufacturer. Produces 150 micron domains; purchase other pin sizes (75&amp;ndash;450 microns) as suited to your particular application.</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr></tbody></table>

a high-throughput cell microarray platform for correlative analysis of cell differentiation and traction forces

微制造蜂窝微阵列，其上的弹性的水凝胶表面由生物分子的接触印刷的组合，用于测量排列生化信号对细胞分化的影响提供一个紧密控制的，高通量的设计系统。采用细胞芯片最近作出的努力证明他们的组合研究中，许多微环境因素的并行呈现效用。然而，这些努力主要集中在调查生化线索对细胞反应的作用。在这里，我们提出了一个细胞芯片平台具有可调的材料特性为牵引力显微镜评估由免疫和生物力学的细胞 - 基底相互作用的两个细胞的分化。要做到这一点，我们已开发利用不同的杨氏模量在任显微镜载玻片或玻璃底培养皿中制成的聚丙烯酰胺水凝胶两种不同格式。我们提供BESŧ实践和故障排除的微阵列对这些水凝胶基质制造，芯片上的后续细胞培养和采集数据。该平台非常适合用在生物过程的调查对其中两个生化（ 例如 ，细胞外基质组合物）和生物物理（ 例如 ，基板的刚度）的线索可能发挥显著，相交的作用。

使用此平台，我们研究在肝脏祖34,35的命运规范生物化学和生物物理线索的作用。 A / G蛋白偶联Notch配体显示出改善的保持和集群中的聚丙烯酰胺水凝胶（ 图3A），并还能够驱动肝祖细胞分化走向胆管细胞命运（ 图3B）。使用单细胞分析，我们量化的Notch配体的ECM蛋白胶原蛋白I，胶原蛋白III，胶原蛋白IV，纤连蛋白，和层粘连蛋白（ 图3C）的反应，发现肝脏祖细胞的配体的反应也取决于ECM上下文。最后，我们利用shRNA的敲除产生肝祖没有配体DLL1和JAG1。向排列Notch配体的反应向变取决于公关任一配体的esence，确认该响应的细胞外配体也是细胞内在配体表达（ 图3D）的功能。此外，我们观察到双阳性的一个独特的亚群（ALB + / OPN +）细胞中的DLL1击倒（ 图3D）。总之，这些代表性的结果表明:（1）阵列格式的组合能力，例示由多个排列ECM蛋白和Notch配体与个别的配位体的击倒的配对; （2）不仅功能排列ECM蛋白，但也可通过蛋白A / G介导的偶联排列的细胞间的配体; （3）我们的单细胞分析和辨别独特的亚群的能力的实现。 我们还观察到肝祖细胞的分化取决于衬底的刚度和ECM组合物（ 图4A上都</str<html>翁&gt;），特别是发现IV型胶原是软，硬衬底支持的分化，而纤连蛋白只支持刚性基板（ 图4B）的分化。 TFM测量的代表热图表明，在对IV型胶原低的衬底的刚度持续牵引应力促进分化成胆管细胞（ 图4C），通过平均根均方值（ 图4D）证实这一发现。总之，这些代表性的结果表明:（1）TFM的成功整合与细胞微阵列上带有可调刚性基板来评估两个细胞表型和牵引应力; （2）同时与基质组合物和基板的刚度肝祖细胞命运的协调;及（3）在细胞微阵列我们的TFM分析和典型的牵引应力分布的执行。 Ë1"SRC ="/文件/ ftp_upload / 55362 / 55362fig1.jpg"/&gt; 图1:概述示意图，显示前三的实验部分。在第一节中，玻璃基板清洗和硅烷化，以促进聚丙烯酰胺水凝胶的制备。在第2节中，感兴趣的生物分子的组合在384孔微孔板源制备。然后，机器人点样仪装有干净引脚，源微孔板和聚丙烯酰胺水凝胶和初始化，制造的水凝胶阵列。在第3节，细胞接种到阵列域和允许粘附，进行兴趣培养协议之后。在终点，细胞是固定的免疫细胞化学/免疫荧光分析或使用TFM。比例尺是75微米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55362/55362fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ="1"&gt; - 页面内<img alt="图2" src="/files/ftp_upload/55362/55362fig2.jpg" /> 图2:处理和阵列免疫荧光数据分析。 （A），瓷砖，复合32位RGB图像首先装箱，然后分割成单独的8位通道。使用排列的荧光标记物和细胞岛的组合，阵列的三个角被确定，以允许自动定向和阵列网格。 （B）中的单细胞数据是为输入阵列的每一个通道产生的。为了解释实验漂移，分位数归一化是通过生物复制施加，产生在所有重复单个共享分布。分位数归一化的数据随后被绘制并经由合奏测量计算解释（ 例如 ，细胞/岛，平均强度，正面为一标签百分比细胞）或单细胞分布的直接分析。M /文件/ ftp_upload / 55362 / 55362fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/55362/55362fig3.jpg" /> 图3:Notch配体介导介绍祖肝脏分化。 （A）的Fc重组Notch配体铁血-1（JAG1）和Delta样1（DLL1）展出时蛋白A / G摆着改进保留和集群。比例尺为50微米。 （二）肝祖细胞在与Notch配体呈现分化成胆管细胞。 4&#39;，6-二脒基-2-苯基吲哚（DAPI）是一种核标签，白蛋白（ALB）是肝细胞标志物，以及骨桥蛋白（OPN）是胆管细胞标记。比例尺为150微米。 （C）细胞百分比阳性OPN上的ECM蛋白的Notch配体JAG1，DLL1和Delta样4（DLL4）的量化胶原蛋白I，Collagen III，IV型胶原，纤连蛋白，层粘连蛋白。学生t -tests分别对每个ECM蛋白中的每个排列Notch配体与P值为P &lt;0.05（*）表示对对照IgG进行。 （D）成像ALB和OPN对胶原III细胞呈现与Notch配体JAG1，DLL1和DLL4流式细胞仪。无缺口肝祖细胞配体DLL1和JAG1（ 即 shDll1和shJag1）用shRNA的敲除产生。在（C）的数据表示为平均值±SEM这个数字已从Kaylan 等改性。 34。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55362/55362fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55362/55362fig4.jpg" /> 图4:基质成分和基质刚度首席运营官rdinate肝祖分化。 （一）肝祖分化为胆管细胞是依赖于这两个ECM成分和基质刚度。 DAPI是核标签，ALB是肝细胞标记，OPN是胆管细胞标志物。 （B）中的正面为OPN细胞百分比上的杨氏模量的基片30千帕，13千帕，并在4kPa用于胶原I（C1）的，IV型胶原（C 4），纤连蛋白（FN），以及所有的双向组合的定量这些细胞外基质蛋白。 （C）细胞牵引压力取决于两个基底刚度和细胞外基质成分上。 （D）上的杨氏模量的基片30千帕和4千帕为胶原蛋白I（C1），IV型胶原（C 4），纤连蛋白（FN），这些的所有双向组合牵引应力的根均方值的量化ECM的蛋白质。在（B）和（D），数据以平均值±SEM和学生t -tests分别针对30千帕执行用于与P值每个的ECM组合为P &lt;0.05（*），P &lt;0.01（**）表示，和P &lt;0.001（***）。比例尺是50微米。这个数字已经从Kourouklis 等进行修改。 35。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55362/55362fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td fo:width="1.2in"> 部分 </td><td fo:width="1.5in"> 问题 </td><td fo:width="1.5in"> 可能原因 </td><td> 解 </td></tr><tr><td rowspan="3"> 1.制造聚丙烯酰胺基质。 </td><td>玻璃罩不能从水凝胶中移除。 </td><td>过聚合。 </td><td>减少聚合时间&lt;10分钟（4瓦/米2）。检查UV crossli北部重点经济区产量预期范围之内。 </td></tr><tr><td>可怜的聚丙烯酰胺水凝胶的聚合。 </td><td> Underpolymerization。 </td><td>增加聚合时间&gt; 10分钟（4瓦/米2）。检查UV交联剂产量预期范围之内。 </td></tr><tr><td>聚丙烯酰胺水凝胶去除玻璃罩后损坏。 </td><td>软聚丙烯酰胺水凝胶是容易损坏。 </td><td>我们观察减少为最柔软的特别（ 即 4千帕）的水凝胶的水凝胶制造的成品率（〜50％）。轻拿轻放的水凝胶，增加的出发人数达到期望的收益率。 </td></tr><tr><td rowspan="2"> 2.制作阵列。 </td><td rowspan="2">可怜的或不一致的斑点形态。 </td><td>加湿器不一致功能。 </td><td>检查加湿器和贯穿每个印数流变仪的功能，并保持65％RH。 </td></tr><tr><td>卡在打印头堵塞或针GED。 </td><td>清洁打印头，允许自由运动的针。干净彻底针或前每个打印运行之后，从销渠道除去聚集。 </td></tr><tr><td rowspan="8"> 3.细胞培养和试验执行。 </td><td rowspan="2">细胞脱落或死亡初始附件后阵列。 </td><td>播和过度增生。 </td><td>降低了初始接种密度和时间。使用数组文化中"维修"或"分化"媒体以减少细胞增殖。 </td></tr><tr><td>从有毒的水凝胶丙烯酰胺单体的释放。 </td><td>水凝胶浸泡在蒸馏水中2 O，至少3天，以允许丙烯酰胺单体的扩散/释放和减少细胞毒性。 </td></tr><tr><td rowspan="4">细胞不重视阵列。 </td><td> Underseeding。 </td><td>增加初始接种密度和时间。使用更强烈粘附的细胞类型。 </td></tr><tr><td>贫困沉积矩阵或生物分子的条件。 </td><td>颗粒和聚集体的清洁销，确认印刷参数，并且评估荧光标记， 例如 ，若丹明共轭葡聚糖斑点。 </td></tr><tr><td>细胞 - 基质相互作用的特异性。 </td><td>不同的细胞类型专门坚持一些而不是其他细胞外基质蛋白。测试多个不同的ECM蛋白与你的细胞。 </td></tr><tr><td>加工后的次优阵列存储。 </td><td>我们建议在65％RH和室温下存放过夜捏造阵列，部分冻结期间避免阶段的变化。细胞粘附是既湿度，温度和存储时间敏感;确保这些参数都是一致的/你的实验进行了优化。 </td></tr><tr><td rowspan="2">细胞培养过程中从玻璃基板的水凝胶的脱离。 </td><td>可怜的滑动清洗和硅烷化。 </td><td>幻灯片清洗更换工作方案和硅烷化。 </td></tr><tr><td> Overdehydrated水凝胶。 </td><td>不离开水凝胶在热板上脱水长于15-30分钟。 </td></tr><tr><td> 4.分析数据。 </td><td>重复点和幻灯片之间的高变异性。 </td><td>变异阵列制造。 </td><td>检查引脚和打印头清洁。确认加湿器的功能。可视化和使用荧光标记量化现货和阵列质量。存储阵列如上建议。 </td></tr></tbody></table> 表1:故障排除。

Watch this Scientific Journal Video about A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces at JoVE.com

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

细胞分化是由微环境因素的影响，包括基质组合物和基材的材料性质的宿主调节。我们在这里描述利用细胞微阵列与牵引力显微镜一起同时评估细胞分化和生物力学细胞 - 基底相互作用的微环境上下文的功能的技术。

细胞分化和牵引力的相关分析高通量细胞芯片平台

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

a-high-throughput-cell-microarray-platform-for-correlative-analysis

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

9.6K 次观看.  University of Illinois at Urbana–Champaign. 细胞分化是由微环境因素的影响，包括基质组合物和基材的材料性质的宿主调节。我们在这里描述利用细胞微阵列与牵引力显微镜一起同时评估细胞分化和生物力学细胞 - 基底相互作用的微环境上下文的功能的技术。 

视频: A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

科研

生物工程

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

一种多功能自动化平台微尺度细胞刺激实验

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

在Micropost阵列方便，准确机械力分析

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

对专为递送治疗因素的神经保护策略成人干细胞高通量表征

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

医学

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

使用 PDMS 的高通量牵引力显微镜揭示了转化生长因子的剂量依赖效应 - 对中皮到中位过渡的影响

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

无参考牵引力显微镜平台的制造和实施

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

牵引显微镜与微流体集成，用于化学集体迁移

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, function at both the cellular and tissue levels,and regulate the mechanical systems in the body. Numerous biological processes are force-dependent, including motility, adhesion, and division of single-cells, as well as contraction and relaxation of organs such as the heart, bladder, lungs, intestines, and uterus. Given its importance in maintaining proper physiological function, cellular contractility can also drive disease processes when exaggerated or disrupted. Asthma, hypertension, preterm labor, fibrotic scarring, and underactive bladder are all examples of mechanically driven disease processes that could potentially be alleviated with proper control of cellular contractile force. Here, we present a comprehensive protocol for utilizing a novel microplate-based contractility assay technology known as fluorescently labeled elastomeric contractible surfaces (FLECS), that provides simplified and intuitive analysis of single-cell contractility in a massively scaled manner. Herein, we provide a step-wise protocol for obtaining two six-point dose-response curves describing the effects of two contractile inhibitors on the contraction of primary human bladder smooth muscle cells in a simple procedure utilizing just a single FLECS assay microplate, to demonstrate proper technique to users of the method. Using FLECS Technology, all researchers with basic biological laboratories and fluorescent microscopy systems gain access to studying this fundamental but difficult-to-quantify functional cell phenotype, effectively lowering the entry barrier into the field of force biology and phenotypic screening of contractile cell force.

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

使用微图案弹性体对单细胞收缩力进行简化的高通量分析

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

微图案牵引显微镜的图案生成

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

用于评估细胞运动和相关信号事件的机械控制的单细胞Durotaxis测定

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...